Glyoxylic acid and other keto acids such as pyruvic acid are important intermediates in the manufacture of various agrochemicals, pharmaceuticals and fragrances. Typical commercial production of glyoxylic acid employs either oxidation chemistry or electrochemistry. Electrochemical manufacture involves either the reduction of oxalic acid or the anodic oxidation of glyoxal to form glyoxylic acid whereas chemical oxidation generally involves the oxidation of glyoxal in the presence of a strong acid such as HNO3. A consequence of these commercial processes is the production of waste streams containing various toxic acids and heavy metals.
Glycolate oxidase is an enzyme available from various sources, including green leafy plants and mammalian cells, which catalyzes the oxidation of glycolic acid to glyoxylic acid, with the concomitant production of hydrogen peroxide. For instance, Tolbert et al., J. Biol. Chem., 181:905 (1949), reported an enzyme, extracted from tobacco leaves, which catalyzed the oxidation of glycolic acid to formic acid and CO2 via the intermediate formation of glyoxylic acid. The addition of certain compounds, such as ethylenediamine, limited the further oxidation of the intermediate glyoxylic acid. The oxidations were carried out at a pH of about 8, typically using glycolic acid concentrations of about 3-40 mM (millimolar). The optimum pH for the glycolate oxidation was reported to be pH 8.9. Oxalic acid (100 mM) was reported to inhibit the catalytic action of the glycolate oxidase. Similarly, Richardson and Tolbert, J. Biol. Chem., 236:1280 (1961), reported the formation of oxalic acid during the glycolate oxidase-catalyzed oxidation of glycolic acid to glyoxylic acid, using enzymes isolated from tobacco, sugar beet, Swiss chard, spinach, or rat liver. Richardson and Tolbert, J. Biol. Chem., 236:1280 (1961), also showed that buffers containing tris(hydroxymethyl)aminomethane (TRIS) inhibited the formation of oxalic acid in the glycolate oxidase catalyzed oxidation of glycolic acid. Clagett et al., J. Biol. Chem., 78:977 (1949) reported that the optimum pH for the glycolate oxidase catalyzed oxidation of glycolic acid with oxygen was about 7.8-8.6, and the optimum temperature was 35°-40° C.
Recent advances in recombinant DNA technology, combined with the isolation of cDNA coding for the spinach glycolate oxidase (see Volokita et al, J. Biol. Chem., 262:15825 (1987)), have allowed for the construction of microbial strains that are intended to serve as alternative, economic enzyme sources. For instance, yeast offers several advantages to commercial applications over Escherichia coli and other bacteria. Yeast can generally be grown to higher densities than bacteria and are readily adaptable to continuous fermentation processing. It has been reported, for example, that Pichia pastoris can be grown to cell densities in excess of 100 g/L (U.S. Pat. No. 4,414,329). Additional advantages of yeast hosts include the fact that many critical functions of the organism, such as oxidative phosphorylations, are located within organelles and thus are not exposed to the possible deleterious effects of the overexpression of foreign enzymatic products. Furthermore, yeasts appear to be capable of glycosylation of expressed polypeptide products, where such glycosylation is important to the bioactivity of the polypeptide product.
Zelitch and Ochoa, J. Biol. Chem., 201:707 (1953), and Robinson et al., J. Biol. Chem., 237:2001 (1962), reported that the formation of formic acid and CO2 in the spinach glycolate oxidase-catalyzed oxidation of glycolic acid resulted from the nonenzymatic reaction of H2O2 with glyoxylic acid. They observed that addition of catalase, an enzyme that catalyzes the decomposition of H2O2, greatly improved the yields of glyoxylic acid by suppressing the formation of formic acid and CO2. With respect to glyoxylic acid production in yeast, glyoxylic acid was produced when glycolic acid and oxygen were reacted in an aqueous solution in the presence of aminomethylphosphonic acid and a catalyst which is a genetically-engineered microbial yeast transformant which expresses the enzyme glycolate oxidase from spinach ((S)-2-hydroxy-acid oxidase, EC 1.1.3.15), and catalase (EC 1.11.1.6) (see U.S. Pat. No. 5,693,490).